(19)
Europaisches Patentamt
European Patent Office
Office europeen des brevets
(11)
EP0 721 015 A1
(12)
EUROPEAN PATENT APPLICATION
published in accordance with Art. 158(3) EPC
(43) Date of publication:
10.07.1996 Bulletin 1996/28
(21) Application number: 94925618.4
(22) Date of filing: 02.09.1994
(51) IntCI 6 : C12P 21/08, C12N5/20,
C12N 15/06, C07K 16/00,
A61 K 39/395
//C12P21:08, C12R1:91
(86) International application number:
PCT/JP94/01453
(87) International publication number:
WO 95/06748 (09.03.1995 Gazette 1995/11)
(84) Designated Contracting States:
AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL
PTSE
(30) Priority: 03.09.1993 J P 242110/93
(71 ) Applicant: CHUGAI SEIYAKU KABUSHIKI KAISHA
Tokyo 115 (JP)
(72) Inventor: FUKUSHIMA, Naoshi
Gotemba-shi, Shizuoka 412 (JP)
(74) Representative: Baillie, lain Cameron
c/o Ladas & Parry
Altheimer Eck 2
80331 Munchen (DE)
(54) MONOCLONAL ANTIBODY THAT INDUCES APOPTOSIS
(57) A monoclonal antibody that induces apoptosis
in the myelocytic cell, a fragment of the antibody, and a
hybridoma that produces the antibody. The antibody is
useful as an antibody that specifically recognizes an anti-
gen or the like inducing apoptosis in the myelocytic cell
and discriminates and identifies the same. Further it is
useable as a useful drug in the field of, for example, treat-
ing myelocytic leukemia by utilizing the activity of induc-
ing apoptosis in the myelocytic cell.
Printed by Rank Xerox (UK) Business Services
2.12.4/3.4
1
Description
Technical Field
The present invention relates to a novel monoclonal 5
antibody having the property of causing apoptosis on
myeloid cells and being useful as medicine for myelocytic
leukemia, and fragments thereof, and furthermore,
relates to a hybridoma producing the monoclonal anti-
body. 10
Since the monoclonal antibodies of the present
invention are useful as antibodies recognizing and iden-
tifying antigens causing apoptosis on myeloid cells spe-
cifically and besides have the property of causing
apoptosis on myeloid cells, they may be used as medi- is
cine useful in the field of remedies for myelocytic leuke-
mia utilizing the property.
Background Art
20
Granulocyte colony-stimulating factors, for example,
recombinant granulocyte colony-stimulating factors (rG-
CSF), have been known primarily as humoral factors to
stimulate the differentiation and proliferation of granulo-
cyte cells, and it has been reported in an experiment 25
upon mice in vivo that the administration of rG-CSF
enhances the hematopoiesis of the bone marrow and in
addition causes remarkable extramedullar hematopoi-
esis in the spleen to proliferate hematopoietic stem cells
and all hematopoietic precursor cells in the spleen. And 30
it has been thought as extramedullar hematopoietic
mechanism in the spleen that hematopoiesis occurs due
to a splenic hematopoietic microenvironment modifica-
tions according to the stimulation of rG-CSF to enhance
hematopoietic potential. 35
Hence, the present inventors have noted splenic
stromal cells administered rG-CSF with a view to clarify-
ing the hematopoietic potential in the spleen, and estab-
lished a hematopoietic stromal cell line (CF-1 cells) from
the spleen of a mouse administered rG-CSF with a view 40
to attempting the analysis of the enhancement of the
hematopoietic potential by stromal cells with rG-CSF,
and examined the potential effect on hematopoiesis
using the hematopoietic stromal cells, and as a result,
colony-stimulating activities in vitro and potency support- 45
ive of hematopoietic stem cells in vivo have been recog-
nized [Blood, 80, 1914 (1992)] .
However, though some of splenic stromal cells have
been established as a cell line (CF-1 cells), and cytolog-
ical characteristics thereof have been examined, no spe- so
cific antibody recognizing surface antigens thereof has
been prepared so far, and characteristics thereof have
been scarcely known yet.
Hence, the present inventors have engaged in assid-
uous studies with a view to developing specific antibod- 55
ies capable of recognizing splenic stromal cells on the
basis of the above information upon splenic stromal cells
and the results of the studies, and prepared monoclonal
antibodies using the splenic stromal cell lines as anti-
2
gens for immunization, and as a result, novel monoclonal
antibodies unreported so far have been obtained.
And as a result of examining the properties of the
obtained monoclonal antibodies, the inventors have
found surprisingly that they have the property of causing
apoptosis on myeloid cells, which has led to the comple-
tion of the present invention.
Disclosure of Invention
It is the objective and purpose of the present inven-
tion to provide a novel monoclonal antibody having the
property of causing apoptosis on myeloid cells and being
useful as medicine for myelocytic leukemia, and frag-
ments thereof, and in addition a hybridoma producing the
monoclonal antibody.
The monoclonal antibody of the present invention is
remarkably useful as an antibody recognizing antigens
causing the apoptosis [ it is also called self-destruction
of cells, phenomenon that a nuclear chromatin DNA is
digested at a nucleosome unit (so-called ladder forma-
tion) to result in the death of cells ] of myeloid cells and
having a function of identifying them or a function of
causing apoptosis on myeloid cells. Incidentally, myeloid
cells include cells other than lymphoid cells, such as neu-
trophils, megakaryocytes, myeloblasts, myelocytes,
mast cells, macrophages, monocytes and erythroblasts,
and the myeloid cells according to the present invention
also mean the same as mentioned above. No mono-
clonal antibody having the property of causing apoptosis
on myeloid cells has been known so far, and hence the
monoclonal antibodies of the present invention are
defined to include all monoclonal antibodies having the
property of causing apoptosis on myeloid cells.
The monoclonal antibody of the present invention
may be prepared basically as stated below.
Namely, the monoclonal antibody of the present
invention may be prepared, for example, by using splenic
stromal cells derived from an animal administered rG-
CSF as antigens, immunizing them according to an ordi-
nary immunization method, cell-fusing the immunized
cells according to an ordinary cell fusion method, and
cloning the fused cells according to an ordinary cloning
method.
As a method of preparing the monoclonal antibody
of the present invention can be preferably exemplified a
method comprising using CF-1 cells, splenic stromal
cells of an animal administered rG-CSF established as
culture cell line by the present inventors, as the antigen
[Blood, Vol. 80, 1 91 4 (1 992)] , fusing plasma cells (immu-
nocyte) of a mammal immunized with the antigen with
myeloma cells of a mammal such as a mouse, cloning
the obtained fused cells (hybridomas), selecting clones
producing antibody according to the present invention
recognizing the above cell line among them, and cultur-
ing them to recover objective antibody. However, the
method is only an example, and in this case, for example,
not only the above CF-1 cells but also cell lines derived
from human splenic stromal cells obtained according to
EP0 721 015 A1
2
3
EP0 721 015 A1
4
the case of CF-1 cells may be used as the antigens prop-
erly to prepare antibodies binding to objective human
myeloid cells in the same manner as in the case of the
above CF-1 cells.
In the method of preparing such monoclonal anti- s
bodies, mammals to be immunized with the above anti-
gen are not particularly restricted; it is preferable to make
selection taking into account suitability with myeloma
cells to be used in cell fusion, and preferably a mouse, a
rat and a hamster are used. 10
Immunization is performed according to an ordinary
method, for example, by administering splenic stromal
cells such as the above CF-1 cells into abdominal cavity
of a mammal by injection. More specifically, it is prefera-
ble to administer one diluted with or suspended in a 15
proper amount of PBS or isotonic sodium chloride solu-
tion to an animal several times every month. It is prefer-
able to use splenic cells removed after the final
administration of the above cells as immunocytes.
As a myeloma cell of a mammal as the other parent 20
cell fused with the above immunocytes can be used pref-
erably known various cells including P3(P3X63Ag8.653)
[J. Immunol., 123, 1548(1978)] , p3-U1 [Current Topics
in Micro-biology and Immunology, 81, 1-7 (1978) ] , NS-
1 [Eur. J. Immunol., 6, 511-519 (1976)] , MPC-11 [Cell, 25
8, 405-415 (1976)] , Sp2/0-Ag14 [Nature, 276, 269-270
(1978)] , FO [J. Immunol. Meth., 35, 1-21 (1980)] , S194
[J. Exp. Med., 148, 313-323 (1978)] and R210 [Nature,
277, 131-133 (1979)] .
The cell fusion of the above immunocyte and a mye- 30
loma cell may be performed basically according to an
ordinary method, for example, a method by Milstein et
al. [Methods Enzymol., 73, 3-46 (1981)] .
More specifically, the above cell fusion may be per-
formed, for example, in an ordinary nutrition medium in 35
the presence of a fusion-accelerating agent. As a fusion-
accelerating agent, polyethylene glycol (PEG) and
Sendai virus (HVJ), and furthermore, adjuvants such as
dimethyl sulfoxide may be added properly if required in
order to enhance the fusing effect. Regarding the ratios 40
of immunocytes and myeloma cells, the former is prefer-
ably used in an amount 1-10 times that of the latter.
Examples of a medium used in the above cell fusion
include a RPMI-1640 medium and a MEM medium suit-
able for the proliferation of the above myeloma cell and 45
other mediums ordinarily used for the culture of this kind
of cell, and in addition, supplementary serum such as
fetal bovine serum (FBS) may be used together.
Cell fusion is performed by mixing prescribed
amounts of the above immunocytes and myeloma cells so
in the above medium, adding a PEG solution preheated
to about 37°C, for example, PEG with an average molec-
ular weight of the order of 1 ,000-6,000 to the medium,
ordinarily, at a concentration of about 30-60 % (W/V),
and mixing them. Subsequently, by repeating the oper- 55
ations of adding proper mediums one after another, cen-
trifuging the reaction mixture and removing the
supernatants can be formed objective hybridomas.
Said hybridomas are selected by culturing in an ordi-
nary selective medium, for example, a HAT medium
(medium supplemented with hypoxanthine, aminopterin
and thymidine). The culture in the HAT medium is con-
tinued for a period sufficient for cells other than objective
hybridomas (non-fused cells) to die out, ordinarily for
several days to several weeks. Subsequently, the
screening and monocloning of the hybridomas produc-
ing the objective antibodies are performed according to
ordinary limiting dilution analysis.
The prepared hybridomas producing the mono-
clonal antibodies of the present invention may be sub-
cultured in an ordinary medium and stored in liquid
nitrogen for a long time.
In order to collect the monoclonal antibodies of the
present invention from the hybridomas may be employed
a method comprising culturing the hybridomas accord-
ing to an ordinary method, and obtaining them from the
supernatants, or a method comprising administering a
hybridoma into a appropriate mammal to proliferate, and
obtaining them from its ascite. The former is suitable for
obtaining antibodies with a high purity and the latter is
suitable for the mass production of antibodies.
Furthermore, the antibodies obtained according to
the above methods may be purified to a high degree
employing an ordinary purification means such as a salt-
ing-out technique, gel filtration and affinity chromatogra-
phy.
The monoclonal antibody of the present invention
may be any one so far as it has a specific property to be
described specifically in Example later, namely, a prop-
erty of causing apoptosis on myeloid cells, and those
having the property are included in the scope of the
present invention, irrespective of the kind of antigens; the
monoclonal antibody of the present invention may be
used as useful medicine for myelocytic leukemia accord-
ing to utilizing the property.
Needless to say, the establishment of a specific sys-
tem for identifying and recognizing antigens causing
apoptosis on myeloid cells according to utilizing the mon-
oclonal antibody of the present invention, or for using it
as medicine for myelocytic leukemia according to utiliz-
ing the specific property thereof, and modification and
application thereof are included within the scope of the
present invention so far as they are put into practice
according to an ordinary method obvious to those skilled
in the art.
Brief Description of Drawings
Fig. 1 shows an analysis (a control in the absence
of an antibody, CF-1 cell) according to immunofluores-
cence.
Fig. 2 shows an analysis of the binding properties of
theGSPST-1 antibody to CF-1 cells according to immun-
ofluorescence.
Fig. 3 shows an analysis of the binding properties of
the BMAP-1 antibody to CF-1 cells according to immun-
ofluorescence.
3
5
EP0 721 015 A1
6
Fig. 4 shows an analysis (a control in the absence
of an antibody, bone marrow cell) according to immun-
ofluorescence.
Fig. 5 shows an analysis of the binding properties of
the GSPST-1 antibody to bone marrow cells according
to immunofluorescence.
Fig. 6 shows an analysis of the binding properties of
the BMAP-1 antibody to bone marrow cells according to
immunofluorescence.
Fig. 7 shows an analysis (a control in the absence
of an antibody, NFS-60) according to immunofluores-
cence.
Fig. 8 shows the binding properties of the GSPST-1
antibody to NFS-60 cells according to immunofluores-
cence.
Fig. 9 shows an analysis (a control according to rat
lgG1, NFS-60) according to immunofluorescence.
Fig. 10 shows the binding properties of the BMAP-
1 antibody to NFS-60 cells according to immunofluores-
cence.
Fig. 1 1 shows an assay for the monoclonal antibody
(BMAP-1) to inhibit NFS-60 cell proliferation.
Fig. 1 2 shows an assay for the monoclonal antibody
(GSPST-1) to inhibit the bone marrow transplantation.
Fig. 1 3 shows an assay for the monoclonal antibody
(BMAP-1) to inhibit the bone marrow transplantation.
Fig. 14 is an explanatory view [microphotograph
(stained with H. E.) of bone marrow samples (x 400)]
showing dead bone marrow cells (2) on 6 days after the
administration of the monoclonal antibody BMAP-1 of
the present invention, and the control (1) in the absence
of the antibody.
Fig. 15 is an explanatory view (migration-photo
according to electrophoretic chromatography) showing
the ladder formation of the DNA of bone marrow cells
observed when the monoclonal antibody BMAP-1 of the
present invention was administered.
Fig. 16 shows a cytotoxicity assay using L929 cells
by TNF a
Fig. 1 7 shows a cytotoxicity assay by the monoclonal
antibody (BMAP-1).
Fig. 1 8 shows an analysis (a control according to rat
lgG2a, BWV1) according to immunofluorescence.
Fig. 19 shows the binding properties of the anti-
mouse MHC class I antibody to BWV1 cells according to
immunofluorescence.
Fig. 20 shows an analysis (a control according to rat
lgG1, BWV1) according to immunofluorescence.
Fig. 21 shows the binding properties of the BMAP-
1 antibody to BWV1 cells according to immunofluores-
cence.
Explanation of Symbols
a: DNA of the thymus of a mouse administered
BMAP-1 (24 hours)
b: DNA of the bone marrow of a mouse administered
BMAP-1 (24 hours)
c: DNA of the bone marrow of a mouse administered
BMAP-1 (8 hours)
d: DNA of the bone marrow of a mouse administered
BMAP-1 (4 hours)
5 e: DNA of the bone marrow of a non-treated mouse
(bone marrow cells)
f : Molecular weight marker
Next, the present invention will be described fur-
10 ther in detail according to Reference Example and
Example, but the present invention is not restricted to the
Example.
Reference Example
15
Establishment of Splenic Stromal Cells and Their Char-
acteristics Thereof
1) Establishment of Splenic Stromal Cells
20
A splenic stromal cell line was established from the
primary culture of the splenic cells of a C57BL76J mouse
administered rG-CSF 100 jug/kg for 5 days. Namely, this
spleen was removed after the administration of rG-CSF
25 under germ-free conditions, cultured in a 25-cm 2 plastic
flask (Corning Co.) for 6 weeks and in an Isocove's mod-
ified Dulbecco's medium (IMDM) (Boehringer-Man-
nheim Co.) with 1 0 % heat-inactivated fetal bovine serum
(FBS) (Sanko Junyaku, Tokyo), 100 U/ml penicillin and
30 1 00 jug/ml streptomycin in an incubator under the condi-
tion of 37 °C and 5 % C0 2 , and the medium was
exchanged for a fresh growth medium twice a week.
In the confluent culture, the adherent cell popula-
tions (stromal cells) were harvested from the flask by
35 using 0.05 % trypsin plus 0.02 % EDTA (Sigma Chemical
Co.) in Ca-,Mg-free PBS, and were transferred into new
flasks. These passages were repeated approximately
once or twice a week. In the early passages (1 st through
1 0th passages), the split ratio of the cells was 1/4 to 1/8,
40 and subsequently the ratio was 1/1 6 to 1/32. The stromal
cells became homogeneous and fibroblastoid after
approximately the 10th passage.
At the 20th passage, these stromal cells were har-
vested as described above and forwarded to cell cloning
45 by using a limiting dilution technique; cell cloning was
repeated twice to establish a stromal cell line (CF-1 cell
line).
Subsequently, these cells were maintained in 5 ml
of IMDM supplemented with 10 % heat-inactivated FBS
so in a 25-cm 2 flask (Corning Co.), and subcultured once
every 5 days at the split ratio of 1/32. Splenic stromal cell
lines can be established from other animals than mouse;
for example, human splenic stromal cell lines can be
established using the same method as described above
55 by transforming the cells with an SV-40 adenovirus vec-
tor [J. Cell. Physiol., 148, 245 (1991) ] .
4
7
EP0 721 015 A1
8
2) Characteristics of CF-1 Cells
CF-1 cells established as a cell line as described
above were examined for alkaline phosphatase, acid
phosphatase, p-glucuronidase, a-naphthyl acetate este- 5
rase and oil red O using standard cytochemical tech-
niques. CF-1 cells were also characterized by
immunoenzymatic histochemistry using the following
monoclonal and polyclonal antibodies: macl (Sero Tec);
factor Vlll-related antigen (Dakopatts); and collagen type 10
I, collagen type III and fibronectin (Chemicon Interna-
tional Inc.). Phagocytosis was tested by latex bead
uptake (particle diameter: 1.09 jum; Sigma), and the
potency of CF-1 cells to convert to adipocytes was tested
by exposure to 10" 6 mol/l hydrocortisone phosphate 15
(Sigma) in a 25-cm 2 flask for 4 weeks after the confluent
culture.
As a result, the CF-1 cells were negative for alkaline
phosphatase, factor Vlll-related antigen, mac I and
phagocytosis, whereas they were positive for collagen 20
type I, collagen type III and fibronectin. CF-1 cells were
not converted to adipocytes during 4 weeks in a conflu-
ent culture with 1 0" 6 mol/l hydrocortisone, although CF-
1 cells had only traces of lipid. From these data, CF-1
cells do not have the characteristics of preadipocytes, 25
macrophages and endothelial cells, and therefore it has
become obvious that they are derived from stromal cells
different from them.
3) Maintenance of Hematopoietic Stem Cells by CF-1 30
Cells
To examine whether hematopoietic stem cells are
maintained by CF-1 cells or not, CFU-S assays (assays
of spleen colonies-forming cells) were performed by the 35
technique of Till and McCulloch. Ten mice per group
were irradiated with 900 cGy (MBR-1520R; Hitachi,
Tokyo) and injected intravenously with bone marrow
mononuclear cells (BM cells) (1.0x 10 5 /head, 5.0 x
10 4 /head, or 2.5 x 10 4 /head) and CF-1 cells (1.0 x 40
10 5 /head), and colonies in the spleen were counted on
the 12th day as CFU-S clones (spleen colonies).
As a result, when bone marrow mononuclear cells
(BM cells) and CF-1 cells were transplanted into irradi-
ated mice, the number of spleen colonies of every group 45
of BM cells increased significantly (between 1.4-1.8
times) as compared to the mice without CF-1 cells, and,
on the 12th day after the transplantation, the survival
ratios of the mice transplanted with BM cells and CF-1
cells were higher than those with only BM cells, showing so
a low death rate; hence it has become apparent that
hematopoietic stem cells are maintained by CF-1 cells.
Best Mode for Carrying Out the Invention
55
The embodiment of the invention will be described
in detail hereinafter.
Example
Establishment of Monoclonal Antibodies
1) Antigens and Immunization
Immunization was performed by using CF-1 cells
obtained in the above Reference Example as antigens.
The cells were cultured in an incubator under the condi-
tion of 5 % C0 2 and 37 °C, using an Isocove's modified
Dulbecco's medium (IMDM) (Boehringer-Mannheim
Co.) supplemented with 10 % fetal bovine serum (FBS;
Sanko Junyaku) as a medium.
The cells were treated with 1mM EDTA/PBS, and
removed from a culture flask according to pipetting. The
cells were suspended into 1mM EDTA/PBS at the cell
number of about 1 x 1 0 7 /ml, and administered to a Wistar
Imamich rat (7-week-old, female; Animal Breeding
Research Laboratory). One ml of cells of about 1x 1 0 7 /ml
were injected into the abdominal cavity of the rat at the
initial immunization, and 1 ml of cells of about 1 x 10 7 /ml
were administered additionally one month later. Further,
1 ml of cells of about 1 x 10 7 /ml were administered addi-
tionally several times at an interval of a month, and after
the reactivity between the immunized rat antibody and
CF-1 cells was recognized, 1 ml of cells of 1 x 10 8 /ml
were administered as the final immunization. Three days
after the final immunization, the rat was killed to remove
spleen.
2) Cell Fusion
After the spleen removed from the rat was minced,
splenic cells isolated were centrifuged, suspended in an
IMDM medium (Boehringer-Mannheim Co.), and
washed intensively. On the other hand, the cells obtained
by cultured mouse myeloma cell line Sp2/0-Ag14
[Nature, 276, 269-270 (1978)] in an IMDM (Boehringer-
Mannheim Co.) supplemented with 10 % fetal bovine
serum (FBS; Sanko Junyaku) were washed in the above
IMDM medium in the same manner, and 1x 10 8 thereof
and 2 x 10 8 of the above splenic cells were put into a
centrifuge tube and mixed to perform cell fusion by pol-
yethylene glycol 4000 (Nakarai Kagaku) according to an
ordinary procedure [Clin. Exp. Immunol., 42 , 458-462
(1 980)] .
Subsequently, the obtained fused cells were dis-
pensed into a 96-well plate with an IMDM medium sup-
plemented with 20 % FBS, and cultured in an incubator
under the condition of 37 °C and 5 % C0 2 . They were
replaced into a HAT selective medium gradually from the
following day, and continued to be cultured.
After the start of the culture, the supernatants were
replaced into a new HAT medium twice a week to con-
tinue the culture and maintain the proliferation.
Next, the obtained fused cells were cloned accord-
ing to an ordinary procedure using limiting dilution anal-
ysis. Namely, only clones having strong binding
properties to antigens were cloned according to an ordi-
5
9
EP0 721 015 A1
10
nary procedure employing limiting dilution analysis by
examining binding properties thereof to the antigens, uti-
lizing antibodies in the supernatants of the above fused
cells.
5
3) Screening
The screening of fused cells (hybridomas) was per-
formed according to indirect fluorescent antibody tech-
nique using flow cytometry. 10
The screening of clones producing objective anti-
bodies was performed using CF-1 cells as target cells.
Namely, cells suspended in a reaction buffer (PBS supp-
plemented with 2 % FBS and 0.02 % NaN 3 ) were cen-
trifuged and recovered as pellets, then suspended in 1 00 is
julI of the hybridoma culture supernatants (about 1 x
10 6 /100 |Lil) and reacted at 4 °C for 1 hour.
After they were washed with the above buffer once, an
FITC-labelled goat anti-rat IgG (FC) antibody (Chemi-
con) was added thereto and incubated for 1 hour. After 20
they were washed once, they were analyzed according
to flow cytometry (FACScan, Becton Dickinson).
4) Purification of Antibodies
25
The fused cells screened in the manner of the above
3) were cultured according to an ordinary procedure, and
antibodies produced in the supernatants were separated
according to an ordinary procedure, and purified.
Namely, hybridomas were recovered from wells with 30
high antibody titers to the antigens, spread in a tissue
culture plastic dish (Corning Co.), cultured under the
condition of 5 % C0 2 and 37 °C, proliferated, and purified
according to an ordinary procedure to obtain monoclonal
antibodies GSPST-1 and BMAP-1 . 35
Regarding GSPST-1, obtained cells were injected
into the abdominal cavity of a BALB/cAJd -nu nude
mouse (8-week-old, male, Nippon Kurea). Produced
ascite was recovered after 10-14 days, salted out with
33 % ammonium sulfate, and dialyzed with PBS. 40
Regarding the BMAP-1 antibody, it was cultured in a
large scale in an Iscove's modified MEM medium sup-
plemented with 10 % FBS, and the supernatants were
concentrated, salted out with 33 % ammonium sulfate,
dialyzed with PBS, purified again by means of a protein 45
A column kit (Amersham), and dialyzed with PBS. Inci-
dentally, in the above Example was described the case
in which the CF-1 cells were used as antigens for immu-
nization; however, it is possible to establish a monoclonal
antibody in the same manner also in case of using other so
stromal cells having potency supportive of hematopoietic
stem cells, and the present invention is not restricted to
the above monoclonal antibodies but includes all mono-
clonal antibodies having the same characteristics and all
hybridomas producing the monoclonal antibodies. 55
A hybridoma producing the monoclonal antibody
BMAP-1 of the present invention is a novel fused cell pre-
pared from a Wistar Imamich rat splenic cell and a mouse
myeloma cell line SP2/0-Ag14 as parent cells, and was
deposited on 9 August, 1 993, under the name of BMAP-
1 (rat mouse hybridoma) with the accession number of
FERM BP-4382, at National Institute of Bioscience and
Human Technology, Agency of Industrial Science and
Technology in Japan [address: 1-3, Higashi 1-chome,
Tsukuba-shi, Ibaraki 305, Japan] , international deposi-
tary authority according to Budapest Treaty on the inter-
national recognition of the deposit of microorganisms for
the purpose of patent procedures.
5) Properties of Antibodies
(i) Reactivity of Antibodies
(Reactivity to CF-1 Cells)
The results of examining the reactivity of the
obtained monoclonal antibodies GSPST-1 and BMAP-1
to CF-1 cells according to immunofluorescence analysis
are shown in Fig. 1 through Fig. 3. Here, Fig. 1 shows
the results of analysis of the control in the absence of an
antibody, Fig. 2 the results of analysis of the binding
properties of GSPST-1 to CF-1 cells, and Fig. 3 the
results of analysis of the binding properties of BMAP-1
to CF-1 cells. In the drawings, vertical axes show relative
number of cells and transverse axes fluorescence inten-
sity.
As is apparent from Fig. 1 through Fig. 3, it has been
revealed that monoclonal antibodies GSPST-1 and
BMAP-1 have properties binding to CF-1 cells and rec-
ognize surface antigens of CF-1 cells.
(Reactivity to Bone Marrow Cells)
Next, the results of analysis of the reactivity of
GSPST-1 and BMAP-1 to normal bone marrow cells
according to flow cytometry (FACScan, Becton Dickin-
son) are shown in Fig. 4 through Fig. 6. Here, Fig. 4
shows the results of analysis of the control in the
absence of an antibody, Fig. 5 the results of analysis of
the binding properties of GSPST-1 to bone marrow cells,
and Fig. 6 the results of analysis of the binding properties
of BMAP-1 to bone marrow cells. In the drawings, vertical
axes show relative number of cells and transverse axes
fluorescence intensity.
As is shown in Fig. 4 through Fig. 6, it has been
revealed that GSPST-1 has not a property binding to
bone marrow cells at all, and that BMAP-1 has a property
binding to all bone marrow cells.
(Reactivity to Myelocytic Leukemic Cell Line (NFS-60))
The results of analysis of the the reactivity of
GSPST-1 and BMAP-1 to NFS-60 cells [Proc. Natl. Acad.
Sci. USA, 82, 6687-6691 (1985)] according to flow
cytometry (FACScan, Becton Dickinson) are shown in
Fig. 7 through Fig. 10. Here, Fig. 7 shows the results of
analysis of the control in the absence of an antibody, Fig.
8 shows the results of analysis of the binding properties
10
15
20
25
6
11
EP0 721 015 A1
12
of GSPST-1 to NFS-60 cells, Fig. 9 shows the results of
analysis of the control using rat lgG1 on the market
(Zymed) and Fig. 1 0 shows the results of analysis of the
binding properties of BMAP-1 to NFS-60 cells. In the
drawings, vertical axes show relative numbers of cells 5
and transverse axes fluorescence intensity.
As is shown in Fig. 7 through Fig. 10, it has been
revealed that GSPST-1 does not react with NFS-60 cells,
and that BMAP-1 has a property binding to NFS-60 cells.
10
(Assay for BMAP-1 to Inhibit Proliferation of NFS-60
Cells)
The results of examining the action of BMAP-1 to
NFS-60 cells in the presence of G-CSF 100 ng/ml and 15
cyclohexyimide 10" 9 M according to the MTT assay
method are shown in Fig. 11. Using culture plates with
96 wells, 10 jutl/well of BMAP-1 solution were added at
concentrations of 0, 10, 100 ng/ml, and 1 , 10, 100 jutg/ml
to 4 x 1 0 3 /well/1 00 jliI of NFS-60 cells, and two days after 20
the numbers of living cells were measured according to
the MTT method. It has been revealed as shown in Fig.
11 that the proliferation of NFS-60 cells is inhibited
remarkably by BMAP-1 .
25
(ii) Typing of Antibodies
Next, as a result of typing the subclass of IgG of the
obtained monoclonal antibodies [using a rat Mono Ab-
ID-Sp kit (Zymed) and a biotin-labelled mouse anti-rat 30
lgG1 antibody (Zymed)] , it has become apparent that
GSPST-1 is lgG2a, and that BMAP-1 is lgG1.
(iii) Potency Inhibiting Bone Marrow Transplantation
35
Next, a test upon the inhibition of bone marrow trans-
plantation was performed using these antibodies to
examine characteristics thereof. The results are shown
in Fig. 12 and Fig. 13. As is shown in Fig. 12 and Fig. 13,
while BMAP-1 has the effect inhibiting the bone marrow 40
transplantation, the effect has not been found in GSPST-
1 . Namely, the above results were obtained by adminis-
tering 1.0 x 10 5 /head of bone marrow cells and mono-
clonal antibodies to C57BL76J mice, irradiated at a fatal
dose of radiation (900 cGy), through the veins of tails, 45
and counting the number of spleen colonies. Incidentally,
"Non-treated" in Fig. 13 shows the case with no admin-
istration of bone marrow cells.
As is shown in Fig. 13, it has been confirmed that it
is because BMAP-1 reacts with bone marrow cells to so
cause apoptosis that the monoclonal antibody inhibits
transplantation completely in the test upon the inhibition
of bone marrow transplantation. Namely, when a hybri-
doma producing BMAP-1 was administered into the
abdominal cavity of a nude mouse, it died at the time 55
when its ascite was stored in a small amount. In addition
it has been revealed that all bone marrow cells died out
according to the intravenous administration of 50
jug/head BMAP-1 to a normal C57BL76J mouse, and in
Fig. 14 is shown a microphotograph backing up the fact
that bone marrow cells on 6 days after the intravenous
administration of BMAP-1 died out. As is apparent from
the microphotograph, it has been observed that not only
lymphoid cells but also neutrophils, megakaryocytes,
myeloblasts, myelocytes, mast cells, macrophages,
monocytes and erythroblasts (so-called myeloid cells)
died out. In addition, as a result of investigating the DNAs
of the bone marrow cells of a mouse administered 30
jmg/head BMAP-1 , apparently ladder formation has been
observed as is shown in Fig. 15, and it has been revealed
that the above reaction of BMAP-1 to bone marrow cells
is due to apoptosis.
The Fc region of the IgG of the BMAP-1 antibody
was digested with pepsin (Sigma) and purified by means
of a GPC column as F(ab' )2, and 33.5 |utg/head (corre-
sponding to 50 jug/head of the whole IgG) were admin-
istered to a C57BL/6J mouse intravenously; as a result
of it, it was observed that bone marrow cells died out in
the bone marrow. It has become apparent according to
the above fact that neither antibody-dependent cell cyto-
toxicity nor complement-dependent cell cytotoxicity par-
ticipates in the cell death of bone marrow cells by BMAP-
1.
As an antigen causing apoptosis has been reported
the Fas antigen of cell surface protein; regarding the Fas
antigen, the expressions of mRNAs of it are recognized
in the thymus, heart, liver, lungs and ovary, but few
mRNAs of it are detected in the bone marrow [J. Immu-
nol., 148, 1274-1279 (1992)] , and hence it is apparent
that antigens recognized by BMAP-1 are different from
the conventionally known Fas antigen.
Furthermore, in order to make it clear whether an
antigen recognized by BMAP-1 would be a TNF receptor
or not, the function of BMAP-1 was investigated using L-
929 cells reacting with TNF to cause cell death. The final
concentrations of a mouse TNF a (Genzyme) were 0,1,
10, 100 pg/ml, 1, 10, 100 ng/ml, and 1 jLtg/ml, and those
of BMAP-1 wereO, 10, 100 pg/ml, 1, 10, 100 ng/ml, and
1,10 jug/ml , and the numbers of living cells of L-929 cells
were measured according to the MTT method on the
second day after the addition of the TNF a and BMAP-
1 . As a result of it, as shown in Fig. 16, Fig. 1 7, while L-
929 cells were reduced by TNF a remarkably, BMAP-1
had no effect upon L-929 cells. Hence, it has become
apparent that an antigen recognized by BMAP-1 is not a
TNF receptor.
The results of investigating whether antigens recog-
nized by BMAP-1 would be MHC class I antigens or not
according to flow cytometry (FACScan, Becton Dickin-
son) are shown in Fig. 18 through Fig. 21. Here, Fig. 18
shows the results of analysis of the control using rat lgG1
(Zymed), Fig. 19 shows the results of analysis of the
binding properties of the anti-mouse MHC class I anti-
body (rat lgG2a, BMA) to BWV1 cells (mouse lymphoma
derived from BW5147 cells), Fig. 20 shows the results
of analysis of the control using rat lgG1 (Zymed) and Fig.
21 shows the results of analysis of the binding properties
of BMAP-1 to BWV1 cells. In the drawings, vertical axes
25
30
35
7
13 EP 0 721 015 A1 14
show relative numbers of cells and transverse axes flu-
orescence intensity. As a result, BMAP-1 did not recog-
nize BWV1 cells but the MHC class I antibody reacted
with BWV1 cells.
As described above, it has been confirmed experi- 5
mentally that BMAP-1 has the function of causing apop-
tosis on myeloid cells; according to the present inventors'
knowledge, no monoclonal antibody having the property
of causing apoptosis on myeloid cells has been reported
so far as described above, and hence monoclonal anti- 10
bodies having such a function are novel ones found by
the present inventors. And, since it is thought that the
monoclonal antibodies of the present invention repre-
sented by BMAP-1 can cause death of myelocytic leuke-
mic cells considered to be high in the expression of 15
antigens thereof by utilizing the function of the apoptosis
of the monoclonal antibody on bone marrow cells, the
monoclonal antibody of the present invention having the
property of causing apoptosis on myeloid cells is useful
as medicine for myelocytic leukemia. 20
The monoclonal antibodies of the present invention
have been described specifically according to the Exam-
ple as above; as the monoclonal antibodies having the
property of causing apoptosis on myeloid cells according
to the present invention may be exemplified those men- 25
tioned as specific examples above, but they are not
always restricted to them but include all monoclonal anti-
bodies having the same characteristic and function pre-
pared in the same manner, irrespective of the kind of
antigens. 30
Industrial Applicability
Since the monoclonal antibodies of the present
invention are useful as antibodies recognizing and iden- 35
tifying antigens causing apoptosis on myeloid cells spe-
cifically and besides have the property of causing
apoptosis on myeloid cells, they may be used as medi-
cine useful in the field of remedies for myelocytic leuke-
mia utilizing the property. 40
Claims
1 . A monoclonal antibody having the property of caus-
ing apoptosis on myeloid cells through binding to the 45
myeloid cells.
2. Fragments of a monoclonal antibody having the
property of causing apoptosis on myeloid cells
through binding to the myeloid cells. so
3. A hybridoma producing the monoclonal antibody
according to Claim 1 .
55
8
EP0 721 015 A1
4 00
FSC
SEC
PL 1
PL 2
S. 25 5
0. 255
□ . 255
•3. 255
PL 1
Fig. 1
500
rsc
ssc
PL 1
PL 2
0. 255
0, 255
0. 255
0. 255
0
m Vn l' iV | ' ii m nil 1 i mini 1 iT i'im
/I
A
10
0
1 0
i
10^
PL 1
T
1 0
10
Pig. 2
EP0 721 015 A1
Pig. 3
500
Fig. 4
10
EP0 721 015 A1
5B0
rsc
ESC
FLl
ri_2
E, 255
B. 255
E» • 25 5
B. 255
FL I
Fig. 5
5Q0
FSC 9,255
SSC B. 25 5
FL.I 9.255
FU2 B.255
FL I
Fig. 6
11
EP0 721 015 A1
0. 255
0. 255
0. 255
0. 255
0
i — rr
I 0
rrrtrj-
I 13
1 — r t
TTTlff
J 0 2
FL 1
1 0 3
T n'TTTTT
1 0
Fig. 7
0. 255
0. 555
0. 255
0. 255
Pig. 8
12
EP0 721 015 A1
□ J u
rsc
ssc
0. 255
0. 255
0. 255
0. 355
0
V.
i — rrnt
i va
0
10 1
t nrrrrj r-"i— rTfTTT| r
° 3
I M 1TTT
10^
FL 1
J 0
10
Pig. 9
rsc
ssc
ri_ i
ri_2
G. 255
0. 255
0. 255
0. 255
Fig. 10
13
EP0 721 015 A1
0.50 -
0.<0 -
CD
0.30 -
CD
O
CD
03
0.20 -
0.10 .-
0.00
stag
: p<0.01
Control 0.9ng/ml 9ngfml 90ng/ml 0.9ng/ml 9ng/ml
Monoclonal Antibody (BMAP-1) Concentration
Assay for BMAP-1 to inhibit NFS-GO cell proliferation
Fig. 11
14
EP0 721 015 A1
14 -i
Com GSPST-i
Monoclonal Antibody
Assay for CSPST-l to inhibit
the bone marrow transplantation
Pig. 12
15
EP0 721 015 A1
p<0.0 (
« ■
6MAP-I
S u g / h
BMAP - I
5 0 \i g / h
N o n - 1
Monoclonal Antibody
Assay for BMAP-1 to inhibit
the bone marrow transplantation
Pig. 13
16
EP0 721 015 A1
( l )
(2 )
Fie. 14
17
EP0 721 015 A1
18
EP0 721 015 A1
1.0 -
<
Conl Ipg lOpglOOpg Ing lOng lOOng \ n q
TNF Concentration ( /ml)
Cytotoxicity assay using L-929 cells by TNF a
Fig. 16
19
EP0 721 015 A1
1.0 n
Conl lOpglOOpg Ing lOnglOOng I \x g iOjiq
BMAP-l Concentration ( /ml)
Cytotoxicity assay using L-929 cells by BMAP-l
Fig. 17
20
EP0 721 015 A1
5 00
rSC O. 255
SSC 0.255
rLI 0.255
R_2 0. 255
FL 1
Fig. 18
5BB
fsc
55C
TL2
0. 255
0. 255
0. 255
0. 255
FL1
Fig. 19
21
EP0 721 015 A1
Fig. 20
513(3
Fig. 21
22
EP0 721 015 A1
INTERNATIONAL SEARCH REPORT
International application No.
PCT/JP94/01453
A. CLASSIFICATION OF SUBJECT MATTER „
Int. CI 6 C12P21/08, C12N5/20 , 15/06, C07K16/00, A61K39/395 //
(C12P21/08, C12Rl:91)
According to International Patent Classification (IP Q or to both national clarification and IPC
B. FTFI DS SEARCHED
Minimum documentation aearched (classification system followed by classification symbols)
Int. Cl5 C12P21/08, C12N5/ 12-5/28 , 15/02-15/90, C07K15/28,
A61K39/395 m
searched other man minimum documents uod to the extent that such documents are included in the fields searched
Electronic data base consulted during the international search (name of date base and, where practicable, search terms used)
WPI, BIOSIS PREVIEWS
C DOCUMENTS CONSIDERED TO BE RELEVANT
Category*
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
Experimental Hematology, Vol. 16, No. 10,
(1988) , Stephen A. Cannistra, et al
"Monocytes enhance gamma- interferon- induced
inhibition of myeloid progenitor cell growth
through secretion of tumor necrosis factor,"
P. 865-870
Neoplasma, Vol. 35, No. 5, (1988),
B. ChorvSth, et al
"Immunocytometric characteristics of
a monoclonal antibody (Bra 55)
recognizing the leukocyte Common antigen
(LCA) , " P. 495-502
1-3
1-3
| | Further documents are listed in the continuation of Box C. | | See patent family annex.
* Special categories of cited documents:
"A" document deflai ag the general slate of the art which is not considered
to be of particular relevance
M E" earlier document but published on or after the international filing dale
"L" document which may throw doubts on priority daimfs) or which is
cited to establish the publication dale of another citation or other
special reason (as specified)
"O" document referring to an oral disclosure, use, exhibition or other
"p* document published prior to the international filing date but later than
the priority date claimed
T* later document published after the international filing date or priority
date and not in conflict with the application but cited to understand
the principle or theory underlying the invention
"X" document of particular relevance; the claimed invention cannot be
considered novel or cannot be considered to involve an inventive
step when the document is taken alone
**Y" document of particular relevance; the claimed invention cannot be
considered to involve an inventive step when the document is
combined with one or more other such documents, such combination
being obvious to a person skilled in the art
"&** document member of the same patent family
Date of the actual completion of the international search
November 16, 1994 (16. 11. 94)
Date of mailing of the international search report 1
December 6, 1994 (06. 12. 94)
Name and mailing address of the ISA/
Japanese Patent Office
Facsimile No.
Authorized officer |
Telephone No.
Form PCT/ISA/210 (second sheet) (July 1992)
23
Live Music Archive
Librivox Free Audio
Metropolitan Museum
Cleveland Museum of Art
Internet Arcade
Console Living Room
Books to Borrow
Open Library
TV News
Understanding 9/11